Postnatal skeletal stem cells in health and disease

Dental &Craniofacial Research

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Abstract

The Section has continued to study post-natal skeletal stem cells (SSCs, also known as "mesenchymal" stem cells) from a variety of tissues, with a particular emphasis on determining the role that they play in the pathophysiology of skeletal diseases, and in particular, in fibrous dysplasia of bone (FD) and the McCune Albright Syndrome (MAS). It has been our hypothesis that any genetic defect (intrinsic factor) or change in the microenvironment (extrinsic factor) that alters the biological activity of skeletal stem cells, which are central mediators of post-natal skeletal metabolism, will cause skeletal abnormalities. Furthermore, based on the remarkable ability of skeletal stem cells to regenerate a complete bone/marrow organ, a major goal is to further develop our techniques for bone regeneration in human patients with skeletal defects. 1. SKELETAL STEM CELLS THAT FORM HARD TISSUES (30% Effort) It has been known since the pioneering work of Alexander Friedenstein, and Maureen Owen and coworkers that the post-natal bone marrow contains a population of non-hematopoietic, adherent and clonogenic stromal cells (Colony Forming Unit-Fibroblasts), a subset of which are self-renewing and multipotent. This subset of ?skeletal stem cells? (SSCs) has the ability to form bone, hematopoietic supportive stroma and marrow adipocytes upon in vivo transplantation with appropriate scaffolds, and to form cartilage in micro-mass cultures. A similar type of cell was found in peripheral blood (the circulating SSC), albeit rarely in humans. Using techniques that we developed for characterizing SSCs from marrow, studies identified stem cells in a number of oro-facial tissues, including dental pulp of deciduous and permanent teeth, and periodontal ligament (reviewed in Gronthos et al., Adult Stem Cells, Gronthos et al., Orthod Craniofac Res). Extensive immunophenotyping and microarray analyses indicate that these different populations share many features, and are undoubtedly related. Yet, upon in vivo transplantation, they form very distinctive types of hard and soft tissues (reviewed in Bianco and Robey; Robey and Bianco, Handbook of Adult Stem Cells, Kuznetzov et al., Cells, In Press). Gaining a better understanding of the biological similarities and differences of this ?family? of skeletal stem cells is of critical importance in deciphering how they maintain normal structure and function of hard tissues, the role that they play in genetic and acquired diseases, and how they can be used in regenerative medicine. Current and future studies will aim to further characterize their similarities and dissimilarities using genomic and proteomic approaches. In addition, they will be compared to human ES cells to determine the factors that regulate self-renewal and subsequent differentiation of the various stem cell populations. 2. THE ROLE OF PARATHYROID HORMONE/PARATHYROID HORMONE RELATED PEPTIDE (PTH/PTHrP) SIGNALLING IN CONTROLLING THE FUNCTION OF SSCs DURING ORGANOGENESIS (10% Effort) PTH and PTHrP bind to a common receptor (PPR) that is expressed at specific sites during skeletal development, and is coupled to Gs-alpha. Consequently, PPR is an upstream member of the same signaling pathway that is affected in fibrous dysplasia of bone, which is caused by activating mutations in Gs-alpha. Recently, a transgenic animal was created in which constitutively active PPR (caPPR) is under the control of the 2.3kb Col1A1 promoter that directs expression specifically in bone and dentin. The caPPR causes over production of cAMP, and thus, these transgenic mice (Col1-caPPR) exhibited a phenotype highly reminiscent of FD. The transgene initially promoted increased bone formation that obliterated marrow spaces, delayed the transition from bone to bone marrow during growth, and retarded the emergence of cell types supporting hematopoiesis and marrow adipocytes. This bone phenotype is very reminiscent of what is observed in FD. With multiple rounds of bone turnover, also characteristic of FD, this phenotype resolved spontaneously over time, leading to the establishment of marrow that could then be assessed for the activity of SSCs. Transgenic marrow contained a greatly reduced number CFU-Fs. While highly proliferative, the progeny of these CFU-Fs were incapable of generating a complete heterotopic bone/marrow organ upon in vivo transplantation into immunocompromised mice, indicating a loss of the SSC subset from the stromal compartment. From these data, it is apparent that appropriate control of PTH/PTHrP signaling and cAMP production is essential for the establishment of a normal bone/marrow organ and for the maintenance of the SSC within it (Kuznetsov et al., J Cell Biol, 2004). 3. FIBROUS DYSPLASIA OF BONE AND THE McCUNE-ALBRIGHT SYNDROME (40% Effort) FD/MAS is a somatic mosaic disease that presents as a spectrum of phenotypes. FD/MAS is caused by post-zygotic activating missense mutations in the G protein, Gs-alpha, at R201 (R201C,H,S,G). These mutations lead to persistent activation of adenylyl cyclase and production of excess cAMP. FD is characterized by focal replacement of bone and marrow with abnormal, osteomalacic bone and fibrous tissue formed by dysfunctional skeletal stem cells. MAS is represented by the clinical triad of FD, cafe-au-lait skin pigmentation and endocrinopathies, all caused by the same mutation, but in tissues derived from different embryonic germ lines. In previous studies, we have shown that the fibrotic tissue is composed of skeletal progenitors. Our current studies are focused on analyzing the biological activity of mutant and normal skeletal stem cells present in FD lesions as a function of aging. The results are quite similar to what we have found for the Col1caPPR mouse, that mutant skeletal stem cells disappear with aging of a lesion. Taken together, these findings suggest that over-activity of cAMP mediated signaling pathways interferes with self-renewal of skeletal stem cells, and leads to their consumption. 4. SKELETAL STEM CELLS IN BONE REGENERATION (20%) Much effort over the last seven years has been dedicated to the development of the techniques to use ex vivo expanded bone marrow stromal cells for bone regeneration in human patients. We are currently preparing another pre-IND application for the FDA that incorporates our methods for ex vivo expansion, the use of hydroxyapatite/tricalcium (HA/TCP) ceramic particles as a scaffolding, and analysis of new bone formation in a canine model. In conjunction, we are developing a clinical protocol for treating patients with cranial defects that have been previously treated by autologous and allogenic bone grafts, bone graft substitutes and surgical plates. In addition to direct orthotopic application of cells attached to HA/TCP particles, we are also developing an injectable construct for percutaneous administration of cells and scaffolding.